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- PDB-7o3d: Cooperation between the intrinsically disordered and ordered regi... -

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Basic information

Entry
Database: PDB / ID: 7o3d
TitleCooperation between the intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly
ComponentsTranscription elongation factor SPT6
KeywordsCHAPERONE / Spt6 / elongation factor / RNA Pol II / nucleosome / CTD
Function / homology
Function and homology information


carbon catabolite repression of transcription from RNA polymerase II promoter by glucose / regulation of transcriptional start site selection at RNA polymerase II promoter / transcription antitermination factor activity, DNA binding / regulation of mRNA 3'-end processing / nucleosome organization / transcription elongation-coupled chromatin remodeling / poly(A)+ mRNA export from nucleus / nucleosome binding / transcription elongation factor complex / transcription antitermination ...carbon catabolite repression of transcription from RNA polymerase II promoter by glucose / regulation of transcriptional start site selection at RNA polymerase II promoter / transcription antitermination factor activity, DNA binding / regulation of mRNA 3'-end processing / nucleosome organization / transcription elongation-coupled chromatin remodeling / poly(A)+ mRNA export from nucleus / nucleosome binding / transcription elongation factor complex / transcription antitermination / transcription elongation by RNA polymerase II / positive regulation of transcription elongation by RNA polymerase II / euchromatin / nucleosome assembly / chromatin organization / histone binding / chromatin remodeling / regulation of transcription by RNA polymerase II / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / mitochondrion / DNA binding / nucleus
Similarity search - Function
: / Spt6, S1/OB domain / YqgF/RNase H-like domain / Likely ribonuclease with RNase H fold. / Spt6 acidic, N-terminal domain / Helix-turn-helix DNA-binding domain of Spt6 / Transcription elongation factor Spt6, YqgF domain / Transcription elongation factor Spt6, helix-hairpin-helix motif / Spt6, SH2 domain, C terminus / Acidic N-terminal SPT6 ...: / Spt6, S1/OB domain / YqgF/RNase H-like domain / Likely ribonuclease with RNase H fold. / Spt6 acidic, N-terminal domain / Helix-turn-helix DNA-binding domain of Spt6 / Transcription elongation factor Spt6, YqgF domain / Transcription elongation factor Spt6, helix-hairpin-helix motif / Spt6, SH2 domain, C terminus / Acidic N-terminal SPT6 / Helix-hairpin-helix motif / Holliday-junction resolvase-like of SPT6 / Helix-turn-helix DNA-binding domain of SPT6 / Tex-like protein, HTH domain superfamily / Tex-like domain superfamily / Spt6, Death-like domain / Transcription elongation factor Spt6 / Spt6, SH2 domain, N terminus / Spt6, SH2 domain / SH2 domain / YqgF/RNase H-like domain superfamily / RuvA domain 2-like / Src homology 2 (SH2) domain profile. / Src homology 2 domains / SH2 domain / SH2 domain superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
Transcription elongation factor SPT6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.71 Å
AuthorsKasiliauskaite, A. / Kubicek, K. / Klumpler, T. / Zanova, M. / Zapletal, D. / Novacek, J. / Stefl, R.
Funding support Czech Republic, 6items
OrganizationGrant numberCountry
Ministry of Education, Youth and Sports of the Czech RepublicLM20115043 Czech Republic
Ministry of Education, Youth and Sports of the Czech RepublicLM20115070 Czech Republic
Ministry of Education, Youth and Sports of the Czech RepublicLM20115042 Czech Republic
European Research Council (ERC)649030 Czech Republic
Ministry of Education, Youth and Sports of the Czech RepublicLQ1601 Czech Republic
Czech Science FoundationGA18-11397S Czech Republic
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Cooperation between intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly.
Authors: Aiste Kasiliauskaite / Karel Kubicek / Tomas Klumpler / Martina Zanova / David Zapletal / Eliska Koutna / Jiri Novacek / Richard Stefl /
Abstract: Transcription elongation factor Spt6 associates with RNA polymerase II (Pol II) and acts as a histone chaperone, which promotes the reassembly of nucleosomes following the passage of Pol II. The ...Transcription elongation factor Spt6 associates with RNA polymerase II (Pol II) and acts as a histone chaperone, which promotes the reassembly of nucleosomes following the passage of Pol II. The precise mechanism of nucleosome reassembly mediated by Spt6 remains unclear. In this study, we used a hybrid approach combining cryo-electron microscopy and small-angle X-ray scattering to visualize the architecture of Spt6 from Saccharomyces cerevisiae. The reconstructed overall architecture of Spt6 reveals not only the core of Spt6, but also its flexible N- and C-termini, which are critical for Spt6's function. We found that the acidic N-terminal region of Spt6 prevents the binding of Spt6 not only to the Pol II CTD and Pol II CTD-linker, but also to pre-formed intact nucleosomes and nucleosomal DNA. The N-terminal region of Spt6 self-associates with the tSH2 domain and the core of Spt6 and thus controls binding to Pol II and nucleosomes. Furthermore, we found that Spt6 promotes the assembly of nucleosomes in vitro. These data indicate that the cooperation between the intrinsically disordered and structured regions of Spt6 regulates nucleosome and Pol II CTD binding, and also nucleosome assembly.
History
DepositionApr 1, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 13, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 15, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transcription elongation factor SPT6


Theoretical massNumber of molelcules
Total (without water)134,9231
Polymers134,9231
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area39810 Å2
MethodPISA

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Components

#1: Protein Transcription elongation factor SPT6 / Chromatin elongation factor SPT6


Mass: 134923.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: the sequence stretches of the sample that do not make part of the coordinates are flexible regions that were not observed in the cryoEM map
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: SPT6, CRE2, SSN20, YGR116W, G6169 / Variant: Wild-type / Production host: Escherichia coli B (bacteria) / Strain (production host): BL21-CodonPlus-RIL / References: UniProt: P23615

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Structure of elongation factor Spt6 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (recombinant)Organism: Escherichia coli B (bacteria)
Buffer solutionpH: 7.5
Buffer componentConc.: 20 mM / Name: HEPES / Formula: HEPES
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: homogeneous monodisperse sample
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / C2 aperture diameter: 30 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 55 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13807
Image scansWidth: 3840 / Height: 3710 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2RELION3.1particle selection
3cryoSPARC3.1particle selection
4crYOLO1.7.5particle selection
12Coot0.9.2model fitting
13cryoSPARC3.1CTF correction
14ISOLDE1.1.0model fitting
16cryoSPARC3.1initial Euler assignment
17cryoSPARC3.1final Euler assignment
18cryoSPARC3.1classification
20cryoSPARC3.13D reconstruction
29PHENIX1.19.2-4158model refinement
30ISOLDE1.1.0model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2995892
Symmetry
IDImage processing-IDEntry-IDPoint symmetry
117O3D
217O3DC1 (asymmetric)
317O3DC1 (asymmetric)
3D reconstructionResolution: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 718639 / Num. of class averages: 313 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficients
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
13PSI

3psi

A11127-1219
23PSF

3psf

A1298-1248
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035875
ELECTRON MICROSCOPYf_angle_d0.7687943
ELECTRON MICROSCOPYf_dihedral_angle_d13.1652230
ELECTRON MICROSCOPYf_chiral_restr0.046878
ELECTRON MICROSCOPYf_plane_restr0.0141038

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